[Invention Defined by a Surface Modication]
Polymers having phosphorylcholine groups have been researched as biocompatible polymers, and biocompatible materials prepared by coating various base agents with such polymers have been developed.
For example, Patent Document 1-1 discloses a cosmetic in which powder coated with a homopolymer or copolymer of 2-methacryloyloxyethyl phosphorylcholine is used as cosmetic powder to improve moisture retention and adhesion on the skin.
Also, Patent Document 1-2 and Patent Document 1-3 disclose medical materials and a separation medium coated with polymers having phosphorylcholine groups.
The surface of the aforementioned materials are coated with a polymer obtained by polymerizing monomers having the phosphorylcholine structure prepared by reacting an acrylic-type monomer mainly having hydroxyl groups with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and then using trimethylamine to turn the reaction product into a quaternary ammonium (refer to Patent Documents 1-4 and 1-5 for the preparation method).
Patent Document 1-4 describes the preparation of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, and Patent Document 1-5 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.    Patent Document 1-1: Japanese Patent Laid-Open H7-118123 bulletin    Patent Document 1-2: Japanese Patent Laid-Open 2000-279512 bulletin    Patent Document 1-3: Japanese Patent Laid-Open 2002-98676 bulletin    Patent Document 1-4: Japanese Patent Laid-Open H9-3132 bulletin    Patent Document 1-5: Japanese Patent Laid-Open H10-298240 bulletin
However, depending on the shape of the material, it is difficult to effectively coat the entire surface of the material by using a method that modifies the surface by coating the material with a polymer having phosphorylcholine groups.
Also, the polymer coating peels off the material's surface and may cause problems in terms of durability. Furthermore, since the material's surface is coated with the polymer, the effects may go beyond the purpose of giving biocompatibility by using phosphorylcholine groups and the basic characteristics required of the material itself may be lost.
Also there is a problem in that the procedure is cumbersome because the aforementioned preparation method for the polymer used for coating requires strict anhydrous conditions. Furthermore, depending on the polymerization conditions, there may be a stability problem for the phosphorylcholine groups bonded to the coating polymer.
In view of the aforementioned description, the inventors conducted earnest research on a method for modifying the surface of various materials by using phosphorylcholine groups, and discovered that a material directly having phosphorylcholine groups on the surface and therefore having superior biocompatibility and hydrophilicity can be obtained with ease and in great versatility by the bond formation reaction on the surface of the material by reacting a compound containing phosphorylcholine groups and a material containing amino groups, rather than coating the material surface with a polymer having phosphorylcholine groups, thus completing the present invention.
The inventors also discovered that, even with a method in which the surface is modified by coating the material with a polymer, application of the method of the present invention described above after a specific polymer coating secures sufficient durability of the phosphorylcholine groups on the material's surface, compared with conventional methods in which the material is simply coated physically by a cast of a hydrophilic polymer. It was also discovered that, depending on the material (materials having a certain thickness made of metal, plastic, glass, etc. such as processed pieces and substrates), the method of the present invention can very easily give hydrophilicity and/or biocompatibility to the material while maintaining the basic properties required of the material, resulting in a material that can be effectively used as a material for a separation analysis apparatus and the like.
[2: Invention Defined by a Modified Powder]
Polymers having phosphorylcholine groups have been researched as biocompatible polymers, and biocompatible materials prepared by coating various base agents with such polymers have been developed.
For example, Patent Document 2-1 discloses a cosmetic in which powder coated with a homopolymer or copolymer of 2-methacryloyloxyethyl phosphorylcholine is used as cosmetic powder to improve moisture retention and adhesion on the skin.
Also, Patent Document 2-2 and Patent Document 2-3 disclose medical materials and a separation medium coated with polymers having phosphorylcholine groups.
The surface of the aforementioned materials are coated with a polymer obtained by polymerizing monomers having the phosphorylcholine structure prepared by reacting an acrylic-type monomer mainly having hydroxyl groups with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and then using trimethylamine to turn the reaction product into a quaternary ammonium (refer to Patent Documents 2-4 and 2-5 for the preparation method).
Patent Document 2-4 describes the preparation of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, and Patent Document 2-5 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.    Patent Document 2-1: Japanese Patent Laid-Open H7-118123 bulletin    Patent Document 2-2: Japanese Patent Laid-Open 2000-279512 bulletin    Patent Document 2-3: Japanese Patent Laid-Open 2002-98676 bulletin    Patent Document 2-4: Japanese Patent Laid-Open H9-3132 bulletin    Patent Document 2-5: Japanese Patent Laid-Open No. H10-298240 bulletin
However it is difficult to effectively coat the entire surface of the powder surface by using a method that coats the surface with a polymer having phosphorylcholine groups. Also, the polymer coating peels off the powder and may cause problems in terms of durability. Furthermore, since the powder's surface is coated with the polymer, the effects may go beyond the purpose of giving biocompatibility by using phosphorylcholine groups and the basic characteristics required of the powder itself may be lost.
Also, there is a problem in that the procedure is cumbersome because the aforementioned preparation method for the polymer used for coating requires strict anhydrous conditions. Furthermore, depending on the polymerization conditions, there may be a stability problem for the phosphorylcholine groups bonded to the coating polymer.
In view of the aforementioned description, the inventors conducted earnest research on various materials having phosphorylcholine groups, and discovered that powder directly having phosphorylcholine groups on the surface can be obtained with ease and with great versatility by the bond formation reaction on the surface of the powder by reacting a compound containing phosphorylcholine groups and powder containing a functional group that reacts with this compound, rather than coating the powder surface with a polymer having phosphorylcholine groups, thus completing the present invention.
[3: Invention Defined by a Chromatography Packing]
Polymers containing phosphorylcholine groups have been researched as biocompatible polymers. Biocompatible materials that are various base agents coated with these polymers have been developed.
For example, Patent Document 3-1 describes medical materials coated with a polymer having phosphorylcholine groups. Also, Patent Document 3-2 describes a separation medium coated with a polymer having phosphorylcholine groups.
The surface of the aforementioned materials are coated with a polymer obtained by polymerizing monomers having the phosphorylcholine structure prepared by reacting an acrylic-type monomer mainly having hydroxyl groups with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and then using trimethylamine to turn the reaction product into a quaternary ammonium (refer to Patent Documents 3-3 and 3-4 for the preparation method).
Patent Document 3-3 describes the preparation of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, and Patent Document 3-4 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.
On the other hand, there are many commercially available packings for GFC, which separates biological samples such as proteins and polypeptides that have smaller molecular weight than proteins by means of size exclusion. For the packings for GFC, there are packings that use cross-linked hydrophilic polymers as the carrier and packings that use silica gel as the carrier.
A packing that uses a cross-linked hydrophilic polymer as the carrier has a wide pH range for the migration phase and is highly versatile. However, compared with a packing that uses silica as the carrier, a packing that uses a polymer as the carrier is: (1) harder to obtain a higher theoretical plate number due to the difficulty of controlling the fine pore size, and (2) often times incapable of obtaining chromatograms with a good reproducibility due to an inferior strength against the high pressure applied when used for high-performance liquid chromatography (HPLC) and the swelling of the particles caused by the migration phase solvent.
A packing that uses silica gel as the carrier has a problem of adsorption of proteins and/or polypeptides onto the surface of the silica gel carrier. To address this problem, there are packings commercially available that use silica gel whose surface is modified with non-dissociative hydrophilic groups for the purpose of suppressing the adsorption of proteins and/or polypeptides in the analysis sample on the silica gel.
For example, Shodex PROTEIN KW-803 (product name) is commercially available from Showa Denko KK as a silica gel-type GFC column. This silica gel-type column is described in the catalogue as a silica gel-type GFC mode column suitable for analyzing proteins having a molecular weight of several thousands to a million.
Also, YMC-Pack Diol (product name) is commercially available from YMC Co. Ltd. This is also described as a silica gel-type GFC column prepared by chemically bonding functional groups having the diol structure to a silica gel carrier; it can be used to separate proteins having a molecular weight of ten thousand to several hundred thousand.    Patent Document 3-1: Japanese Patent Laid-Open 2000-279512 bulletin    Patent Document 3-2: Japanese Patent Laid-Open 2002-98676 bulletin    Patent Document 3-3: Japanese Patent Laid-Open H9-3132 bulletin    Patent Document 3-4: Japanese Patent Laid-Open H10-298240 bulletin
The object of the present invention is to provide a new packing for chromatography. When the packing for chromatography of the present invention is used for a GFC column, the adsorption of protein and/or polypeptides is very small and a higher separation ability is demonstrated.
[4: Invention Defined by a Liquid Contact Member]
A liquid-contacting member of piping of an analytical apparatus and the like is designed to prevent protein adsorption by means of coating with a common hydrophilic group. For example, Patent Document 4-1 describes a capillary column that is hard for proteins to adsorb onto, allows higher separation and higher reliability analysis, and is designed to have a longer life by means of the formation of a polymer film on the inside wall using a vinyl compound. Patent Document 4-2 describes an electrophoresis capillary tube that allows easy removal of proteins and has a long service life; this capillary tube has agarose bonded covalently to the inside wall of a capillary tube made of a silica base material. The description claims that successful separation of proteins with good reproducibility is achieved without degrading the separation efficiency and the service life can be extended. Patent Document 4-3 proposes apparatuses and materials for clinical testing in which adhesion/adsorption of proteins and platelets in blood is controlled; specifically, it discloses a plastic polypropylene Spitz tube whose surface is coated with a hydrophilic polymer such as vinyl alcohol.
Also, polymers having phosphorylcholine groups have been researched as biocompatible polymers, and biocompatible materials prepared by coating various base agents with such polymers have been developed. For example, Patent Document 4-4 and Patent Document 4-5 disclose medical materials and a separation medium coated with polymers having phosphorylcholine groups. These are prepared by synthesizing monomers having the phosphorylcholine structure and then coating the surface with the polymer obtained by polymerizing these monomers. For a method for manufacturing these polymers, Patent Document 4-6 describes the preparation of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, and Patent Document 4-7 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.    Patent Document 4-1: Japanese Patent Laid-Open H5-288716 bulletin    Patent Document 4-2: Japanese Patent Laid-Open H6-288984 bulletin    Patent Document 4-3: Japanese Patent Laid-Open S62-169052 bulletin    Patent Document 4-4: Japanese Patent Laid-Open 2000-279512 bulletin    Patent Document 4-5: Japanese Patent Laid-Open 2002-98676 bulletin    Patent Document 4-6: Japanese Patent Laid-Open H9-3132 bulletin    Patent Document 4-7: Japanese Patent Laid-Open H10-298240 bulletin
The object of the present invention is to provide a liquid-contacting member coated with a polymer having hydrophilic groups; it provides a liquid-contacting member much more firmly coated with a hydrophilic polymer compared with a liquid-contacting member prepared by simply coating physically the surface of piping and such of an analytical apparatus by a cast of a hydrophilic polymer. Furthermore, the hydrophilic group is a phosphorylcholine group, which has a high protein adsorption prevention effect, and therefore a liquid-contacting member that allows protein analysis with a good reproducibility and a high recovery rate is provided.
Depending on the shape of the liquid-contacting member, effective or even coating on the entire inner wall surface is difficult when conventional technology is used wherein polymer having phosphorylcholine groups is obtained first and then this polymer is used to coat the liquid-contacting member to modify the surface.
Also, the polymer coating peels off the material's surface and may cause problems in terms of durability. There is also a problem in that the procedure is cumbersome because the monomer having a phosphorylcholine group has to be prepared under strict anhydrous conditions. Furthermore, depending on the polymerization conditions for this monomer, there is a stability problem for the phosphorylcholine groups bonded to the coating polymer.
In view of the description above, the present invention provides a method that is not cumbersome and gives superior stability of the phosphorylcholine group. That is, the present invention is a liquid-contacting member on whose surface phosphorylcholine groups are introduced by first carrying out very firm polymer coating using a polymer containing alkoxysilyl groups on the inner wall surface of the liquid-contacting member and then bonding a chemical compound containing phosphorylcholine groups, rather than coating the inner wall surface of the liquid-contacting member with a polymer prepared by polymerizing monomers having phosphorylcholine groups. Furthermore, the present invention is not selective about the choice of the material of the liquid-contacting member; it is highly versatile and can be applied to liquid-contacting members composed of materials such as metal, plastic, and glass.
[5: Invention Defined by a Filter]
The separation process using separation membranes has broadened its application range as industries developed and now it encompasses a wide range including production of ultra-pure water for the semiconductor industry, removal of molds in the food industry, separation of oil and emulsion in the chemical industry, and concentration, separation, and purification of physiologically active substances in the medical drug industry.
Particularly in recent years, separation and purification of proteins and the like in the biochemical field has become important for the prospering bioindustry; and membranes having a higher fractionation efficiency and superior durability are in demand.
The methods to separate proteins are largely divided into separation by precipitation, separation by adsorption, and separation in solutions; separation by adsorption uses various kinds of chromatography, the separation in solutions uses gel filtration, electrophoresis, liquid-phase distribution method, membrane separation method, etc.; of these, the membrane separation method is known to be a method that quickly and easily separates proteins.
The membrane separation method is generally classified, by the size of the target substance for separation, into the precise filtration method, the ultrafiltration method, the dialysis method, the electrodialysis method, the reverse osmosis method, and the gas separation method. Their definitions according to the IUPAC are as follows: Microfiltration (acronym MF): a process in which particles and polymers larger than 0.1 micrometers are blocked; ultrafiltration (UF): a process in which particles and polymers within the range of 0.1 micrometers to 2 nanometers are blocked; nanofiltration (NF): a process in which particles and polymers smaller than 2 nanometers are blocked; reverse osmosis (RO): a process in which, due to the applied pressure, the solvent migrates in the direction opposite to the osmotic pressure difference.
Conventional filter materials have a problem in that proteins, oils/fats, and the like were adsorbed on the membrane surface and the filter's separation characteristics, particularly the membrane transport flow rate, decrease (refer to Nonpatent Document 1).
As the target substance for separation becomes smaller, the filter precision becomes more important; with conventional separation membranes, it is very difficult to separate between proteins with similar sizes due to the limitation of fractionation characteristics and such of the membrane.
Polymers containing phosphorylcholine groups have been researched as biocompatible polymers. Biocompatible materials that are various base agents coated with these polymers have been developed.
Patent Document 5-1 describes the preparation of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylate, and Patent Document 5-2 describes the preparation of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine.
Patent Document 5-3 describes medical materials coated with a polymer having phosphorylcholine groups.
Patent Document 5-4 discloses a separation medium coated with a polymer having phosphorylcholine groups.
The surface of the aforementioned materials are coated with a polymer obtained by polymerizing monomers having the phosphorylcholine structure prepared by reacting an acrylic-type monomer mainly having hydroxyl groups with 2-chloro-1,3,2-dioxaphosphorane-2-oxide and then using trimethylamine to turn the reaction product into a quaternary ammonium (refer to Patent Documents 5-1 and 5-2 for the preparation method).
This method simply coats the surface physically with a polymer having the phosphorylcholine structure and therefore the evenness and durability of the coating film is hardly sufficient.
Also, in order to improve the fractionation characteristics, it is desirable for the filter to have homogeneous pores controlled at the nano level.
A known example of inorganic materials that have homogeneous pores controlled at the nano level is porous alumina prepared by the anode oxidation of aluminum wherein the anode oxidation conditions are strictly controlled in an acidic bath.
Non-patent Document 5-2 reports formation of an alumina structure having homogeneous pores in the range of 5-500 nm by using sulfuric acid, oxalic acid, and phosphoric acid for the acidic electrolysis bath and controlling the anode oxidation voltage according to the type of the acid.
As described above, there have been various researches aimed at controlling the adsorption of proteins on the filter; however, it has been limited in terms of the filter materials, pore size and such, and no filter useful for wider applications has been developed yet.    Patent Document 5-1: Japanese Patent Laid-Open H9-3132 bulletin    Patent Document 5-2: Japanese Patent Laid-Open H10-298240 bulletin    Patent Document 5-3: Japanese Patent Laid-Open H2000-279512 bulletin    Patent Document 5-4: Japanese Patent Laid-Open 2002-98676 bulletin    Non-patent document 5-1: Nakagaki, Masayuki, ed., Membrane Treatment Technology System, vol. 1 of 2, Fuji Techno System, p 259    Non-patent document 5-2: Masuda, Hideki, Chemistry and Education, Vol. 47-8 (1999), p 520
The object of the present invention is to provide a new filter material. Also, the present invention discloses a method for manufacturing a filter for which protein adsorption is suppressed. The filter material of the present invention is useful for separation and concentration of antibodies, enzymes and the like, as well as for filtration of a wide range of biological substances, e.g. filters for blood dialysis.
According to the present invention, filter materials with very little adsorption of proteins and polypeptides can be provided.